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Biosynthesis
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7-Dehydrocholesterol is the precursor of cholecalciferol. Within the epidermal layer of skin, 7-Dehydrocholesterol undergoes an electrocyclic reaction as a result of UVB light at wavelengths between 290 and 315 nm, with peak synthesis occurring between 295 and 300 nm. This results in the opening of the vitamin precursor B-ring through a conrotatory pathway making previtamin D3 (pre-cholecalciferol). In a process which is independent of UV light, the pre-cholecalciferol then undergoes a [1,7] antarafacial sigmatropic rearrangement and therein finally isomerizes to form vitamin D3.

The active UVB wavelengths are present in sunlight, and sufficient amounts of cholecalciferol can be produced with moderate exposure of the skin, depending on the strength of the sun. Time of day, season and altitude affect the strength of the sun, and pollution or glass all affect the production. Exposure of face, arms and legs, averaging 5–30 minutes twice per week, The darker the skin, and the weaker the sunlight, the more minutes of exposure are needed. Vitamin D overdose is impossible from UV exposure; the skin reaches an equilibrium where the vitamin degrades as fast as it is created.

The light emitted by the UV lamps in tanning beds (which produce ultraviolet primarily in the UVA spectrum, but typically produce 4% to 10% of the total UV emissions as UVB).

It can be disputed whether cholecalciferol and all forms of vitamin D are by definition "vitamins", since the definition of vitamins includes that the substance cannot be synthesized by the body and must be ingested; cholecalciferol is synthesized by the body during UVB radiation exposure.

The three steps in the synthesis and activation of vitamin D3 are regulated as follows:
 * Cholecalciferol is synthesized in the skin from 7-dehydrocholesterol under the action of ultraviolet B (UVB) light. It reaches an equilibrium after several minutes depending on the intensity of the UVB in the sunlight - determined by latitude, season, cloud cover, and altitude - and the age and degree of pigmentation of the skin.
 * Hydroxylation in the endoplasmic reticulum of liver hepatocytes of cholecalciferol to calcifediol (25-hydroxycholecalciferol) by 25-hydroxylase is loosely regulated, if at all, and blood levels of this molecule largely reflect the amount of cholecalciferol produced in the skin combined with any vitamin D2 or D3 ingested.
 * Hydroxylation in the kidneys of calcifediol to calcitriol by 1-alpha-hydroxylase is tightly regulated: it is stimulated by parathyroid hormone and serves as the major control point in the production of the active circulating hormone calcitriol (1,25-dihydroxyvitamin D3).